scholarly journals Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasuaki Kohama ◽  
Shuichiro Higo ◽  
Yuki Masumura ◽  
Mikio Shiba ◽  
Takumi Kondo ◽  
...  
Keyword(s):  
Direct Injection ◽  
Adeno Associated Virus ◽  
Systemic Injection ◽  
Guide Rna ◽  
Human Cardiomyocytes ◽  
Wide Range ◽  
Targeted Integration ◽  
Induced Pluripotent

Abstract Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

scholarly journals LncRNA ZNF593-AS Alleviates Contractile Dysfunction in Dilated Cardiomyopathy

2021 ◽  
Author(s):  
Jiahui Fan ◽  
Huaping Li ◽  
Rong Xie ◽  
Xudong Zhang ◽  
Xiang Nie ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Contractile Function ◽  
Treatment Strategies ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Human Cardiomyocytes ◽  
Underlying Mechanisms ◽  
Imaging Gene Expression

Rationale: Previously, we identified the human cardiac long non-coding RNAs (lncRNAs) profile in dilated cardiomyopathy (DCM) patients, among which ZNF593-AS, also named as RP11-96L14.7 and ENST00000448923.2, showed good conservation among species. Objective: We aim to elucidate the mechanism underlying lncRNA in DCM and DCM that lead to heart failure, which might provide new insights into the mechanisms of DCM and possible treatment strategies in the future. Methods and Results: lncRNA expression was measured by real-time PCR and in situ hybridization assays. Coding potential was verified by bioinformatic and biologic assays. Recombinant adeno-associated virus with cardiac specific promoter was used to deliver lncRNA in vivo, while cardiac structure and functions were assessed by echocardiography and catheter. Sarcomere shortening, calcium imaging, gene expression profiling, and pull-down assays were performed to investigate the underlying mechanisms. ZNF593-AS, which mainly localized in the cytoplasm of cardiomyocytes, was robustly decreased in the failing heart of DCM patients, as well as in phenylephrine-treated human cardiomyocytes. Overexpression of mmu-ZNF593-AS significantly improved transverse aortic constriction (TAC)-induced cardiac dysfunction in mice. Moreover, ZNF593-AS overexpression restored the aberrant Ca 2+ handling and contractility of cardiomyocytes from TAC-treated mice. Further, we found that ZNF593-AS acted as a guide RNA scaffold and recruited HNRNPC to ryanodine receptor type 2 (RYR2) mRNA, which in turn facilitated RYR2 mRNA stability, contributed to the improvement of cardiac Ca 2+ handling and contractile function in DCM. Conclusions: Our findings suggested that lncRNA-based therapeutics may protect against DCM.

scholarly journals CRISPR/Cas9-mediated gene editing induces neurological recovery in an A53T-SNCA overexpression rat model of Parkinson’s disease

2020 ◽  
Author(s):  
Hyung Ho Yoon ◽  
Sunghyeok Ye ◽  
Sunhwa Lim ◽  
Seung Eun Lee ◽  
Soo-Jin Oh ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Rat Model ◽  
Therapeutic Potential ◽  
Negative Control ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Dopaminergic Neurodegeneration

AbstractTo date, no publicly available disease-modifying therapy for Parkinson’s disease has been developed. This can be partly attributed to the absence of techniques for in vivo deletion of the SNCA gene (encoding α-synuclein), which is one of the key players in Parkinson’s disease pathology. In particular, A53T-mutated SNCA (A53T-SNCA) is one of the most studied familial pathologic mutations in Parkinson’s disease. Here we utilized a recently discovered genome editing technique, CRISPR/Cas9, to delete A53T-SNCA in vitro and in vivo. Among various CRISPR/Cas9 systems, SaCas9-KKH with a single guide RNA (sgRNA) targeting A53T-SNCA was packaged into adeno-associated virus. Adeno-associated virus carrying SaCas9-KKH significantly reduced A53T-SNCA levels in A53T-SNCA-overexpressed HEK293T cells, without off-target effects on wild-type SNCA. Furthermore, we tested the technique’s in vivo therapeutic potential in a viral A53T-SNCA overexpression rat model of Parkinson’s disease. Gene deletion of A53T-SNCA significantly prevented the overexpression of α-synuclein, dopaminergic neurodegeneration, and parkinsonian motor symptoms, whereas a negative control without sgRNA did not. Our findings propose CRISPR/Cas9 system as a potential therapeutic tool for A53T-SNCA familial Parkinson’s disease.

scholarly journals An Alternate Route for Adeno-associated Virus (AAV) Entry Independent of AAV Receptor

2018 ◽  
Vol 92 (7) ◽  
Author(s):  
Amanda M. Dudek ◽  
Sirika Pillay ◽  
Andreas S. Puschnik ◽  
Claude M. Nagamine ◽  
Fang Cheng ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vector ◽  
Cell Attachment ◽  
Vector System ◽  
Large Set ◽  
Adeno Associated Virus ◽  
Systemic Injection ◽  
Entry Pathway

ABSTRACTDeterminants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well asin silico-reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observedin vivo, as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, bothin vitroandin vivo, suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy.IMPORTANCEAdeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.

Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

1978 ◽  
Vol 36 (2) ◽  
pp. 650-651
Author(s):  
Raul I. Garcia ◽  
Evelyn A. Flynn ◽  
George Szabo
Keyword(s):  
Direct Injection ◽  
Skin Pigmentation ◽  
Freeze Fracture ◽  
Pigment Granule ◽  
Time Lapse ◽  
Ultrastructural Level ◽  
Lanthanum Tracer ◽  
A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

Ethnomedicinal uses, Phytochemistry and Pharmacological Aspects of the Genus Berberis Linn: A Comprehensive Review

2020 ◽  
Vol 23 ◽  
Author(s):  
Roohi Mohi-ud-din ◽  
Reyaz Hassan Mir ◽  
Prince Ahad Mir ◽  
Saeema Farooq ◽  
Syed Naiem Raza ◽  
...  
Keyword(s):  
Chemical Constituents ◽  
Pharmacological Activities ◽  
Traditional Use ◽  
Comprehensive Review ◽  
Wide Range ◽  
Anti Cancer ◽  
Comprehensive Survey ◽  
Ethnomedicinal Uses

Background: Genus Berberis (family Berberidaceae), which contains about 650 species and 17 genera worldwide, has been used in folklore and various traditional medicine systems. Berberis Linn. is the most established group among genera with around 450-500 species across the world. This comprehensive review will not only help researchers for further evaluation but also provide substantial information for future exploitation of species to develop novel herbal formulations. Objective: The present review is focussed to summarize and collect the updated review of information of Genus Berberis species reported to date regarding their ethnomedicinal information, chemical constituents, traditional/folklore use, and reported pharmacological activities on more than 40 species of Berberis. Conclusion: A comprehensive survey of the literature reveals that various species of the genus possess various phytoconstituents mainly alkaloids, flavonoid based compounds isolated from different parts of a plant with a wide range of pharmacological activities. So far, many pharmacological activities like anti-cancer, anti-hyperlipidemic, hepatoprotective, immunomodulatory, anti-inflammatory both in vitro & in vivo and clinical study of different extracts/isolated compounds of different species of Berberis have been reported, proving their importance as a medicinal plant and claiming their traditional use.

Current Status and Future Perspective for Research on Medicinal Plants with Anticancerous Activity and Minimum Cytotoxic Value

2019 ◽  
Vol 20 (12) ◽  
pp. 1227-1243
Author(s):  
Hina Qamar ◽  
Sumbul Rehman ◽  
D.K. Chauhan
Keyword(s):  
Side Effects ◽  
Medicinal Plants ◽  
Anticancer Agents ◽  
Drug Targets ◽  
Psidium Guajava ◽  
Current Status ◽  
Future Perspective ◽  
Wide Range

Cancer is the second leading cause of morbidity and mortality worldwide. Although chemotherapy and radiotherapy enhance the survival rate of cancerous patients but they have several acute toxic effects. Therefore, there is a need to search for new anticancer agents having better efficacy and lesser side effects. In this regard, herbal treatment is found to be a safe method for treating and preventing cancer. Here, an attempt has been made to screen some less explored medicinal plants like Ammania baccifera, Asclepias curassavica, Azadarichta indica, Butea monosperma, Croton tiglium, Hedera nepalensis, Jatropha curcas, Momordica charantia, Moringa oleifera, Psidium guajava, etc. having potent anticancer activity with minimum cytotoxic value (IC50 >3μM) and lesser or negligible toxicity. They are rich in active phytochemicals with a wide range of drug targets. In this study, these medicinal plants were evaluated for dose-dependent cytotoxicological studies via in vitro MTT assay and in vivo tumor models along with some more plants which are reported to have IC50 value in the range of 0.019-0.528 mg/ml. The findings indicate that these plants inhibit tumor growth by their antiproliferative, pro-apoptotic, anti-metastatic and anti-angiogenic molecular targets. They are widely used because of their easy availability, affordable price and having no or sometimes minimal side effects. This review provides a baseline for the discovery of anticancer drugs from medicinal plants having minimum cytotoxic value with minimal side effects and establishment of their analogues for the welfare of mankind.

Approach in improving potency and selectivity of kinase inhibitors: Allosteric kinase inhibitors

2020 ◽  
Vol 21 ◽  
Author(s):  
Shangfei Wei ◽  
Tianming Zhao ◽  
Jie Wang ◽  
Xin Zhai
Keyword(s):  
Protein Interactions ◽  
Kinase Inhibitors ◽  
Binding Modes ◽  
Allosteric Inhibitors ◽  
Wide Range ◽  
Allosteric Sites ◽  
Protein Functions ◽  
Regulate Protein

: Allostery is an efficient and particular regulatory mechanism to regulate protein functions. Different from conserved orthosteric sites, allosteric sites have distinctive functional mechanism to form the complex regulatory network. In drug discovery, kinase inhibitors targeting the allosteric pockets have received extensive attention for the advantages of high selectivity and low toxicity. The approval of trametinib as the first allosteric inhibitor validated that allosteric inhibitors could be used as effective therapeutic drugs for treatment of diseases. To date, a wide range of allosteric inhibitors have been identified. In this perspective, we outline different binding modes and potential advantages of allosteric inhibitors. In the meantime, the research processes of typical and novel allosteric inhibitors are described briefly in terms of structureactivity relationships, ligand-protein interactions and in vitro and in vivo activity. Additionally, challenges as well as opportunities are presented.

In Silico Assessment of ADME Properties: Advances in Caco-2 Cell Monolayer Permeability Modeling

2019 ◽  
Vol 18 (26) ◽  
pp. 2209-2229 ◽  
Author(s):  
Hai Pham-The ◽  
Miguel Á. Cabrera-Pérez ◽  
Nguyen-Hai Nam ◽  
Juan A. Castillo-Garit ◽  
Bakhtiyor Rasulev ◽  
...  
Keyword(s):  
Intestinal Absorption ◽  
In Silico ◽  
Review Paper ◽  
Cell Monolayer ◽  
Cell Permeability ◽  
Drug Substances ◽  
Wide Range ◽  
Modeling Techniques

One of the main goals of in silico Caco-2 cell permeability models is to identify those drug substances with high intestinal absorption in human (HIA). For more than a decade, several in silico Caco-2 models have been made, applying a wide range of modeling techniques; nevertheless, their capacity for intestinal absorption extrapolation is still doubtful. There are three main problems related to the modest capacity of obtained models, including the existence of inter- and/or intra-laboratory variability of recollected data, the influence of the metabolism mechanism, and the inconsistent in vitro-in vivo correlation (IVIVC) of Caco-2 cell permeability. This review paper intends to sum up the recent advances and limitations of current modeling approaches, and revealed some possible solutions to improve the applicability of in silico Caco-2 permeability models for absorption property profiling, taking into account the above-mentioned issues.

Pleiotropic Effect of Mahanine and Girinimbine Analogs: Anticancer Mechanism and its Therapeutic Versatility

2019 ◽  
Vol 18 (14) ◽  
pp. 1983-1990 ◽  
Author(s):  
V. Lenin Maruthanila ◽  
Ramakrishnan Elancheran ◽  
Ajaikumar B. Kunnumakkar ◽  
Senthamaraikannan Kabilan ◽  
Jibon Kotoky
Keyword(s):  
Biological Effects ◽  
Pleiotropic Effect ◽  
In Vivo Evaluation ◽  
Chemopreventive Agents ◽  
Cycle Arrest ◽  
Wide Range ◽  
Anticancer Mechanism ◽  
Metastasis Development

Emerging evidence present credible support in favour of the potential role of mahanine and girinimbine. Non-toxic herbal carbazole alkaloids occur in the edible part of Murraya koenigii, Micromelum minutum, M. zeylanicum, and M. euchrestiolia. Mahanine and girinimbine are the major potent compounds from these species. In fact, they interfered with tumour expansion and metastasis development through down-regulation of apoptotic and antiapoptotic protein, also involved in the stimulation of cell cycle arrest. Consequently, these compounds were well proven for the in-vitro and in vivo evaluation that could be developed as novel agents either alone or as an adjuvant to conventional therapeutics. Therefore, mahanine and girinimbine analogs have the potential to be the promising chemopreventive agents for the tumour recurrence and the treatment of human malignancies. In this review, an updated wide-range of pleiotropic anticancer and biological effects induction by mahanine and girinimbine against cancer cells were deeply summarized.

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